RESUMO
Aha1 is a ubiquitous cochaperone of the Hsp90/Hsp70 chaperone machine. It binds the middle domain of Hsp90 and stimulates ATPase activity, suggesting a function late in the chaperone pathway. Saccharomyces Mal63 MAL activator is a DNA-binding transcription factor and Hsp90 client protein. This study utilizes several MAL activator mutants to investigate Aha1 function in vivo. Deletion of AHA1 enhances induced Mal63-dependent maltase activity levels 2-fold, whereas overproduction of Aha1 represses expression. Maltase expression in strains carrying constitutive and super-inducible mutant activators with alterations near the C terminus (particularly residues 433-463) is unaffected by either aha1Delta or Aha1 overproduction. However, another constitutive activator with alterations outside of this C-terminal region is sensitive to Aha1 regulation. Previously, we showed that in the absence of inducer, Mal63 forms a stable intermediate complex with Hsp70, Hsp90, and Sti1, whereas noninducible mutant activators bind only with Hsp70 in an apparent early complex. Here, we find that triple Myc-tagged Aha1/Myc3 copurifies with all noninducible Mal63 mutant activators tested. Aha1/Myc3 association with inducible Mal63 is observed only in a sti1Delta strain, in which Hsp90 binding and intermediate complex formation are defective. Constitutive and super-inducible mutant activators with C-terminal alterations do not bind Aha1 even in a sti1Delta strain. Mal63 binding to Hsp90 and Hsp70 is enhanced 3-fold by loss of Aha1. These results suggest an interaction between Aha1 and residues near the C terminus of Mal63 thereby regulating Hsp90 association. A novel mechanism for the negative regulation of the MAL activator by Aha1 cochaperone is proposed.
Assuntos
Adenosina Trifosfatases/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Chaperoninas/genética , Deleção de Genes , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Regulação Fúngica da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Complexos Multiproteicos/genética , Ligação Proteica/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Fatores de Transcrição/genética , alfa-Glucosidases/biossíntese , alfa-Glucosidases/genéticaRESUMO
The Hsp90/Hsp70 chaperone machine is an essential regulator of cell growth and division. It is required for activation of select client proteins, chiefly protein kinases and transcription activators and thus plays a major role in regulating intracellular signaling and gene expression. This report demonstrates, in vivo, the association of the Saccharomyces cerevisiae maltose-responsive transcription activator Mal63 (MAL-activator) with the yeast Hsp70 (Ssa1), Hsp90 (Hsp82), and Hop (Sti1) homologs, using a collection of inducible, constitutive, and noninducible alleles. Each class of mutant activator forms a distinctly different stable multichaperone complex in the absence of maltose. Inducible Mal63p associates with Ssa1, Hsp82, and Sti1 and is released in the presence of maltose. Noninducible mal63 mutant proteins bind to Ssa1 alone and do not stably associate with Hsp82 or Sti1. Constitutive MAL-activators bind well to Hsp82 and poorly to Ssa1 and Sti1, but deletion of STI1 restores Ssa1 binding. Taken together, Mal63p regulation requires the formation of Hsp90/Hsp70 subcomplexes comparable to, yet distinct from those observed with previously characterized Hsp90 clients including glucocorticoid receptor and yeast Hap1p. Thus, comparative studies of different client proteins highlight functional diversity in the operation of the Hsp90/Hsp70 chaperone machine.
